[RASMB] re: RASMB digest, Vol 1 #196 - 5 msgs
John Champagne
john.champagne at wyatt.com
Fri Jun 27 13:58:00 PDT 2003
Arthur,
I agree to your explanation with one exception. I believe that Ewa used a Wyatt DAWN EOS to measure the static light scattering signal. At 690nm (which is the wavelength that is used in the instrument) a protein with a Rh of 4.2nm measured by dynamic light scattering, would act as an isotropic scatterer. Therefore you could not possibly measure a Rg by means of static light scattering. If you could measure the Rg and the Rh for this molecule, the ratio of the two measurements would give you information about the mass distribution.
John
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Today's Topics:
1. (no subject) (Claus Urbanke)
2. Re: frctional ratio (Arthur Rowe)
3. Re: frctional ratio (Peter Schuck)
4. Re: frctional ratio (Arthur Rowe)
5. Re: frctional ratio (Peter Schuck)
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Message: 1
From: "Claus Urbanke" <urbanke.claus at mh-hannover.de>
To: <rasmb at rasmb-email.bbri.org>
Date: Fri, 27 Jun 2003 07:23:01 +0200
Subject: [RASMB] (no subject)
Hi all,
unfortunately I broke my tool with which I cut window gaskets for XL-A
analytical cells. It was once (30 years ago) delivered together with a
Spinco Model E. Does anyone out there know how or where to get a
replacement?
Thanks
Claus Urbanke
Dr. Claus Urbanke
Medizinische Hochschule
Biophysikalisch-Biochemische Verfahren
OE 8830
Carl Neuberg Str. 1
30625 Hannover
Tel. (49) 511 532 9372/3707
Fax (49) 511 532 5966
Email Urbanke at bpc.mh-hannover.de
--__--__--
Message: 2
Date: Fri, 27 Jun 2003 11:16:53 +0100
Subject: Re: [RASMB] frctional ratio
From: Arthur Rowe <arthur.rowe at nottingham.ac.uk>
To: Ewa Folta-Stogniew <stogniew at BIOMED.MED.YALE.EDU>,
<rasmb at rasmb-email.bbri.org>
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Dear Ewa
A spherical, unhydrated protein of mass 41.7 kD would have an expected Rh =
2.3 nm (if a vbar = 0.73 ml/g is assumed). That means a frictional ratio =
4.2/2.3 = 1.83.
As you have correctly inferred, that is clearly not a simple globular
protein, for which a frictional ratio in the region of 1.1-1.2 is expected.
There are two possibilities:
(1) you have one of these 'natively unfolded' proteins, whose normal state
is a loosely structured swollen one, or
(2) you have an elongated protein, ball-park axial ratio of around 10 giving
a Perrin function (that part of the frictional ratio which can be attributed
to shape rather than hydration/swelling) = around 1.5 (derived from
1.83/1.2)
If you have done static light LS, you ought to be able to extract an Rg
value (you can download a pdf about Rg values etc from
rabbit.cbe.uiowa.edu/Regression.pdf) from the slope of the Zimm plot.
Unfortunately, the difference between the two models is not great, and SLS
does not tend to give values of the precision you can get with SANS or SAXS.
If - and its a big IF - you have enough material and the kit to hand to do
an intrinsic viscosity and an s upon c plot, you are home and dry, as the
Ratio Function ks/[eta] has a value of 1.6 for the swollen spherical model,
and half that value for an elongated model.
But, that is quite a bit more work. To be honest, on the basis of the data
you have to date, you might as well toss a coin. Only hint I can give is
that natively unfolded proteins of low-ish molecular mass are pretty unusual
things. See Uversky, V.N. (2002) Eur J Biochem 269 2-12. So - if I was
putting money it, I would go for an elongated shape !
All best wishes for your work
Arthur Rowe
--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD UK
Tel: +44 (0)115 951 6156
+44 (0)116 271 4502
Fax: +44 (0)115 951 6157
email: arthur.rowe at nottingham.ac.uk
arthur.rowe at connectfree.co.uk (home)
Web: www.nottingham.ac.uk/ncmh/business
*******************************************************
I had just analyzed a protein which has MW of 41.4 kDa from static LS
analysis (monomer 41.7kDa) and Rh=4.2nm from dynamic LS.
What can I infer about the shape (beyond that it is not globular)?
Pointing to references appreciated.
Thank you, Ewa
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<HTML>
<HEAD>
<TITLE>Re: [RASMB] frctional ratio</TITLE>
</HEAD>
<BODY>
<BLOCKQUOTE><FONT COLOR=3D"#800000">Dear Ewa<BR>
<BR>
A spherical, unhydrated protein of mass 41.7 kD would have an expected Rh =3D=
2.3 nm (if a vbar =3D 0.73 ml/g is assumed). That means a frictional ratio =3D =
4.2/2.3 =3D <U>1.83</U>. <BR>
<BR>
As you have correctly inferred, that is clearly not a simple globular prote=
in, for which a frictional ratio in the region of 1.1-1.2 is expected. There=
are two possibilities:<BR>
<BR>
(1) you have one of these 'natively unfolded' proteins, whose normal state =
is a loosely structured swollen one, <I>or<BR>
</I><BR>
(2) you have an elongated protein, ball-park axial ratio of around 10 givin=
g a Perrin function (that part of the frictional ratio which can be attribut=
ed to shape rather than hydration/swelling) =3D around 1.5 (derived from 1.83/=
1.2)<BR>
<BR>
If you have done static light LS, you ought to be able to extract an Rg val=
ue (you can download a pdf about Rg values etc from rabbit.cbe.uiowa.edu/Reg=
ression.pdf) from the slope of the Zimm plot. Unfortunately, the difference =
between the two models is not great, and SLS does not tend to give values of=
the precision you can get with SANS or SAXS. <BR>
<BR>
If - and its a big IF - you have enough material and the kit to hand to do =
an intrinsic viscosity <I>and</I> an s upon c plot, you are home and dry, as=
the Ratio Function ks/[eta] has a value of 1.6 for the swollen spherical mo=
del, and half that value for an elongated model. <BR>
<BR>
But, that is quite a bit more work. To be honest, on the basis of the data =
you have to date, you might as well toss a coin. Only hint I can give is tha=
t natively unfolded proteins of low-ish molecular mass are pretty unusual th=
ings. See Uversky, V.N. (2002) Eur J Biochem <B>269</B> 2-12. So - if I was =
putting money it, I would go for an elongated shape !<BR>
<BR>
All best wishes for your work<BR>
<BR>
Arthur Rowe<BR>
<BR>
<BR>
</FONT></BLOCKQUOTE>--<BR>
*******************************************************<BR>
Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
NCMH Business Centre<BR>
University of Nottingham<BR>
School of Biosciences<BR>
Sutton Bonington<BR>
Leicestershire LE12 5RD UK<BR>
<BR>
Tel: +44 (0)115 951 6156<BR>
+4=
4 (0)116 271 4502<BR>
Fax: +44 (0)115 951 6157<BR>
email: arthur.rowe at nottingham.ac.uk<BR>
ar=
thur.rowe at connectfree.co.uk (home)<BR>
Web: www.nottingham.ac.uk/ncmh/bu=
siness<BR>
*******************************************************<BR>
<BR>
<FONT COLOR=3D"#800000"><BR>
</FONT><BLOCKQUOTE><FONT COLOR=3D"#800000"><BR>
<TT><BR>
<BR>
</TT></FONT><TT><BR>
I had just analyzed a protein which has MW of 41.4 kDa from static LS<BR>
analysis (monomer 41.7kDa) and Rh=3D4.2nm from dynamic LS.<BR>
<BR>
What can I infer about the shape (beyond that it is not globular)?<BR>
Pointing to references appreciated. <BR>
<BR>
Thank you, Ewa<BR>
<BR>
_______________________________________________<BR>
RASMB mailing list<BR>
RASMB at rasmb-email.bbri.org<BR>
http://rasmb-email.bbri.org/mailman/listinfo/rasmb<BR>
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</TT>
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Message: 3
Date: Fri, 27 Jun 2003 08:25:53 -0400
To: <rasmb at rasmb-email.bbri.org>
From: Peter Schuck <pschuck at helix.nih.gov>
Subject: Re: [RASMB] frctional ratio
Ewa and Arthur,
there's also a possibility that the frictional ratio could be quite high if
the protein is glycosylated. In my experience, that can increase the
friction quite a bit.
Peter
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Message: 4
Date: Fri, 27 Jun 2003 13:40:16 +0100
Subject: Re: [RASMB] frctional ratio
From: Arthur Rowe <arthur.rowe at nottingham.ac.uk>
To: Peter Schuck <pschuck at helix.nih.gov>, <rasmb at rasmb-email.bbri.org>
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Peter -
Agreed, glyco stuff can be 'floppy', and increase friction. However, in the
present case, given that the mass estimated by SLS (41.4 kD) agrees well
with (what I take to be) the formula weight (41.7 kD), it is not too likely
to be an explanation, is it ?
Arthur
Ewa and Arthur,
there's also a possibility that the frictional ratio could be quite high if
the protein is glycosylated. In my experience, that can increase the
friction quite a bit.
Peter
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<BLOCKQUOTE>Peter -<BR>
<BR>
Agreed, glyco stuff can be 'floppy', and increase friction. However, in the=
present case, given that the mass estimated by SLS (41.4 kD) agrees well wi=
th (what I take to be) the formula weight (41.7 kD), it is not too likely to=
be an explanation, is it ?<BR>
<BR>
Arthur<BR>
<TT><BR>
Ewa and Arthur,<BR>
there's also a possibility that the frictional ratio could be quite high if=
<BR>
the protein is glycosylated. In my experience, that can increase the =
<BR>
friction quite a bit.<BR>
Peter<BR>
<BR>
_______________________________________________<BR>
RASMB mailing list<BR>
RASMB at rasmb-email.bbri.org<BR>
http://rasmb-email.bbri.org/mailman/listinfo/rasmb<BR>
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Message: 5
Date: Fri, 27 Jun 2003 08:43:38 -0400
To: <rasmb at rasmb-email.bbri.org>
From: Peter Schuck <pschuck at helix.nih.gov>
Subject: Re: [RASMB] frctional ratio
Arthur,
in that case, of course you're right. I thought the mass was maybe from
mass spectrometry.
Peter
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End of RASMB Digest
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==============================
John Champagne
Applications Scientist \ Regional Manager
Wyatt Technology Corporation
30 South La Patera Lane, B-7
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Tel. (805)681-9009 x121
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